Cultivation and recovery of micro-organisms



United States Patent 3,536,585 CULTIVATION AND RECOVERY OF MICRO-ORGANISMS Bernard Maurice Laine, Lavera, Bouches-du-Rhone, France, assignor to The British Petroleum Company Limited, London, England, a corporation of England No Drawing. Filed Jan. 25, 1967, Ser. No. 611,561 Claims priority, application Great Britain, Feb. 3, 1966,

6 Int. Cl. A23j 1/18; C12c 11/08 US. Cl. 195-28 18 Claims ABSTRACT OF THE DISCLOSURE Aerobic cultivation of a micro-organism on a hydrocarbon feedstock consisting in part of straight chain hydrocarbons thereafter separating from the product, through the use of a surfactant, a fraction containing, as the major proportion thereof, unconsumed hydrocarbons together with a minor proportion of water and a minor proportion of surfactant, heating said fraction, whereby the surfactant is rendered partially or substantially water-insluble, separating a hydrocarbon phase containing surfactant from an aqueous phase and hydrogenating the hydrocarbon phase under conditions such that the surfactant is rendered less effective or ineffective as an emulsifying agent.

This invention relates to a process for the removal of straight chain hydrocarbons, wholly or in part, from a hydrocarbon mixture and to the production of a purified hydrocarbon fraction therefrom.

This invention also relates to a process as hereinbefore described with growth and recovery of a micro-organism. 7

According to the present invention there is provided a process which comprises cultivating a micro-organism which is capable of growing on at least some straight chain hydrocarbons, cultivation being carried out in the presence of a hydrocarbon feedstock which consists in part of straight chain hydrocarbons in the presence of an aqueous nutrient medium and in the presence of a gas containing free oxygen and thereafter separating from the cultivated mixture, through the use of a surfactant, a fraction containing, as the major proportion thereof, unconsumed hydrocarbons together with a minor proportion of water and a minor proportion of surfactant, heating said fraction, whereby the surfactant is rendered partially or substantially water-insoluble, separating a hydrocarbon phase containing surfactant from an aqueous phase and hydrogenating said hydrocarbon phase under conditions such that the surfactant is rendered less effective or ineffective as an emulsifying agent.

We have found that by the operation of the process described a hydrocarbon fraction is obtained which, notwithstanding the use of a surfactant in its recovery, is substantially free of agents which would lead to emulsification or foaming in the event that the hydrocarbon product is either intentionally or fortuitously mixed with an aqueous medium. When a water soluble surfactant is employed in the hydrocarbon recovery stage the separation of the bulk of the aqueous phase from the hydrocarbon phase is much assisted but then remains a problem of breaking an emulsion of hydrocarbon and water which is also recovered. We have found that this emulsion can be effectively broken by heating; however, in consequence the surfactant is transferred to the hydrocarbon phase. By the overall process, which includes a hydrogenation stage, the modified surfactant is reduced in effectiveness or is rendered ineffective.

The process of the invention, including optional stages, will now be described in further detail:

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stocks may be unrefined or may have undergone some refinery treatment, but must contain a proportion of straight chain hydrocarbons in order to fulfil the purpose of this invention. Suitably the petroleum fraction will contain 345% by weight of straight chain hydrocarbons.

The process of the invention is of particular value for the treatment of petroleum gas oil fractions which contain straight chain hydrocarbons in the form of waxes, since by the process of the invention a gas oil of improved pour point is obtained while the waxes are converted to a valuable product.

Within the term micro-organism used herein we include mixtures of micro-organisms. Preferably the micro-organism is capable of growing on at least some normal parafiins.

Micro-organisms which are cultivated as herein described may be yeasts, moulds or bacteria.

7 The yeasts in this specification are classified according to the classification system outlined in The Yeasts, a Toxonomic Study by J. Lodder and N. J. W. Kreger- Van Rij, published by North Holland Publishing Co. (Amsterdam) (1952).

The bacteria mentioned in this specification are classified according to the classification system outlined in Bergeys Manual of Determinative Bacteriology by R. S. Breed, C. G. D. Murray and N. R. Smith, published by Bailliere, Tindall and Cox (London), 7th edition (1957).

Preferably when a yeast is employed this is of the family Cryptococcoaceae and particularly of the sub-family Cryptococcocideae however, if desired there may be used, for example, ascosporogeneous yeasts of the subfamily Saccharomycoideae. Preferred genera of the Cryptococcocideae sub-family are Torulopsis (also known as Torula) and Candida. Preferred strains of yeast are as follows. In particular it is preferred to use the specific stock of indicated Baarne reference number; these reference numbers refer to CBS stock held by the Central Bureau vor Schimmelculture, Baarne, Holland and to INRA stock held by the Institut National de la Recherche Agronomique, Paris France.

Candida lipolytica Candida pulcherrima CBS 610 Candida utilis Candida uzilis, Variati major CBS 841 Candida tropicalis CBS 2317 Torulopsis colliculosa CBS 133 Hansenula anomala CBS Oidium lactis Neurosporo sitophila Mycoderma cancoillote INRA: STV 11 Of the above Candida lipolytica is particularly preferred.

If desired the micro-organism may be a mould. Suitable moulds are Penicillium and preferably there is used peill cillillm expansum. Another suitable genus is Aspergil us.

If desired the micro-organism may be a bacterium. Suitably the bacteria are of one of the orders: Pseudomonadales, Eubacteriales and Actinomycetales.

Preferably the bacteria which are employed are of the families Corynebacteriaceae, Micrococoaceae, Achromobacteraceae, Actincymycetaceae, Rhizobiaceae, Bacillaceae and Pseudomonadaceae. Preferred species are Bacillus megaterium, Bacillus subtilis and Pseudomonas aeruginosa. Other strains which may be employed include:

X anthomonas begoniae Flavobacterium devorans Acetobacter sp. Actinomyces sp. Nocardia opaca The growth operation will usually be discontinued before the stationary phase. If desired the growth stage may be continuous.

After the growth stage it will usually be possible to separate the micro-organism, contaminated with some unmetabolised feedstock and aqueous nutrient medium, from the bulk of the unmetabolised feedstock fraction. Preferably the separation is achieved by means of a decantation; additionally or alternatively centrifuging may be used. The fraction containing the micro-organism is now subjected to treatment with an aqueous treating medium comprising water, a metal salt and a surface active agent consisting of or containing a non-ionic detergent. Preferably the treatment is carried out using one or more of the process conditions as hereinafter described.

The product recovered from the fermenter is decanted to separate a fraction, consisting of about two thirds by volume of the product and consisting mainly of spent nutrient medium, from a product fraction containing substantially all of the product micro-organism and of the residual hydrocarbon together with some spent nutrient medium. To the product fraction is added a mixture of equal volumes of sea water and fresh water, this mixture being added in an amount such that there is obtained a resultant mixture containing about gmS./litre of mineral salts. To this resultant mixture is added a nonionic surface active agent and the resultant mixture is centrifuged, suitably in a Sharples DG2 machine.

Preferably the mixture is centrifuged at 2535 C., for example at about 30 C.

Preferably the non-ionic detergent comprises, in the molecule, a chain of ethylene oxide groups. Preferably the detergent has the formula A(CH CH O)H where A is an alcohol residual group or acid residual group, the compound HA being selected from the following alcohols and acids and the value of n lying in a range as hereinafter shown, the range varying according to the compound HA, selected:

Range of average HA value of n Lauric alcohol 710 Myristic alcohol 7.5-11 Oleic alcohol 13-15 Palmitic acid l4-17 Oleic acid 8.5-11 Stearic acid 15.5-19

As an alternative to the above preferred detergents there may be used a detergent obtained by condensing ethylene oxide with a mixture of lauric alcohol and myristic alcohol to form a product having an ethylene oxide chain of an average 2 to 10 ethylene oxide groups per terminal group; more particularly it is preferred that the value within this range is 8.5.

The fraction containing the micro-organism is obtained from the centrifuge as a paste or cream; this fraction is washed with fresh water and again centrifuged.

The fraction containing the micro-organism so obtained may be treated either by (a) Drying, suitably by spray drying or drum drying and then extracted with an azeotropic mixture of hexane and alcohol, or

(b) Extracted with a mixture of hexane and alcohol and then dried, suitably by spray drying or drum drying.

Preferred methods for use in the cultivation of the micro-organism and for recovery of the product are clescribed in French specification 1,393,517 and, where appropriate, methods or process conditions therein described may be employed in the process of the present invention.

By centrifuging the product from the fermenter (preferably after decanting) there is also obtained an oil phase containing as an emulsion some water and surface active agent.

This oil phase is heated whereby the surface active agent is transferred from the water phase to the oil phase and the emulsion is broken. Preferably the oil phase is heated above the cloud point of the surfactant in water. Preferably a temperature in the range 100 C. is employed.

Without substantial cooling of the product thus obtained (which would lead to the re-establishment of the emulsion) the product is subjected to phase separation.

The oil phase which is recovered contains surfactant which, if retained, would have a deleterious effect on the properties of the oil since, in the presence of water (which might be introduced intentionally or accidentally), the oil would have foaming characteristics. The oil phase is subjected to hydrogenation under conditions such that the surfactant is rendered less effective or ineffective as an emulsifying agent.

Suitable catalysts for use in the hydrogenation stage are compounds of cobalt and molybdenum with or without iron, nickel metal, nickel/tungsten sulphide, or any other conventional hydrogenation or desulphurising catalyst.

Preferably hydrogenation will be carried out under the following conditions.

The temperature may lie in the range -500 C., usually 300-500 C., according to the catalyst; pressure from 10 70 kgs./ sq. cm.; space velocity from 1-10 vol./ vol/hour, hydrogen/hydrocarbon ratio in the range 0.1/1 to 5/1. The hydrogenation stage may be carried out in liquid, gas or mixed phase.

Preferred methods for use in the cultivation of the micro-organism and for the recovery of the product are described in British Patent Specification No. 914,567- also in the following British patent applications, U.S. applications and US. patents:

US. Patent 3,257,289, US. patent application Ser. No. 249,012 (filed Jan. 2, 1963, now abandoned); US. patent application Ser. No. 330,522 (filed Dec. 16, 1963, now abandoned); US. patent application Ser. No. 330,523 (filed Dec. 16, 1963, now abandoned); US. Pat. 3,271,266; US. Pat. 3,258,406; US. Pat. 3,259,549; U.S. Pat. 3,264,196; U.K. patent application 49,060/62; U.K. patent application 49,061/62; U.K. patent application 49,062/62; US. patent application Ser. No. 330,529 (filed Dec. 16, 1963 and now abandoned); UK. patent application 2,234/ 63; U.K. patent application 7,623/ 63; U.K. patent application 19,271/ 63; U.K. patent application 20,803/ 6 3; U.K. patent application 20,804/ 63; U.K. patent application 21,253/63; US. patent 3,268,419; U.K. patent application 38,942/63; U.K. patent application 45,005/63; US. Pat. 3,268,414; U.K. patent application 27,284/ 65; U.K. patent application 44,388/65; U.K. patent application 4,763/66; U.K. patent application 4,765/ 66; U.K. patent application 5,640/ 66 also in the Specifications of French patent application N0s.: 924,254, 925,327.

The invention is illustrated but not limited with reference to the following Examples 1-3.

EXAMPLE 1 40 litres of an aqueous mineral nutrient medium having the composition given below were introduced in a stainless steel fermenter having an effective capacity of 60 litres.

In order to keep the temperature in the fermenter constant at 30 C., water was circulated in an annulus constituted by the space between two concentrical cylinders, the smaller one being the fermenter itself.

The aqueous nutrient medium had the composition:

Distilled water add. 1000 ml.

20 litres of a 24 hours inoculum of Candida tropicalis on mixed C -C normal hydrocarbons were then added, such that the cellular density was about 1 gram of dry matter per litre.

1,030 litres of heavy gas oil, that is grams/litre, were then added this quantity being sufiicient to take the cellular density to 2 grams/ litre.

The temperature of the culture was kept at 30i1 C. pH at 4 and aeration and agitation giving 3 millimoles of 0 per litre of medium per minute. Ammonia solution was admitted by an automatic pH controller.

When the flow of ammonia reached ml. the addition of gas oil was begun assuming a yield on gas oil of dry yeast produced gas oil feed of 10% by weight and a cell division time of 3 hours. This addition was carried out every hour until 200 grams/litre, i.e. 13.8 litres had been added.

Starting with a cellular density of 2 grams/litre, at

The trace amount of detergent was found to have led to undesirable emulsifying characteristics in the gas oil.

This gas oil was tested for emulsion forming characteristics by the test method ASTM B.1400.01 56T. In this test is used ccs. of water, 8 cc. of kerosine (as diluent) and 32 ccs. of the gas oil undergoing test. Results were as shown in the following Table 1.

The recorded gas oil containing trace amounts of water and detergent was heated to 90 C., whereby the detergent was dissolved in the oil phase, and centrifuged at 90 C. in a Sharples tubular centrifuge at 14,000 g. to remove water.

The residual gas oil so obtained was hydrogenated in the presence of a cobalt molybdate catalyst containing:

Percent M00 12.6 by weight C00 2.45 by weight 200 ccs. of the catalyst was used in the form of 2.5 mm. x 2.5 mm. cylindrical pellets and hydrogenation conditions were:

Temperature395 C. Pressure-40 kg.

Throughput of hydrogen and gas oil are shown in the following Table.

The treated gas oil was tested for emulsion forming characteristics by the test method ASTM B.1400-01 56 T. In this test is used 40 cc. of water, 8 cc. of kerosine (as diluent) and 32 ccs. of the gas oil undergoing test. Re-

hours (at the end of the exponential growth phase) the 35 Sults were as shown in the following Table 1.

TABLE 1 Run 1 Run 2 Run 3 Hydrogenation conditions:

Gas oil throughput rate vol./

vol./hour 3 5 10 Hydrogen throughput rate litres lhour) 85 180 Hydrogenated gas oll Emulsification Feed Recovered test stock 1 gas 011 Run 1 Run 2 Run 3 13sec. sec. 74 sec. 1 39 41 48 27 40 38 30 Emulsion (co) 52 2 2 2 1 Kerosine diluted.

cellular density was 15 grams/litre. The fermenter was EXAMPLE 2 Grams .per litre Spent mineral medium 839 Non-metabolized gas oil 112 Paste of micro-organism 49 The non-metabolized gas oil had the following characteristics:

Sp. gr. 60 (60 F.): 0.8803 r1 1.490 Sulphur, percent by wt.: 1.84 Cloud point: +1 C. Pour point: 6 C. Water: Trace Detergent: Trace 40 litres of an aqueous mineral medium having the following composition were introduced into a stainless steel fermenter having an effective capacity of 60 litres; the composition of the aqueous nutrient medum was:

Grams Diammonium hydrogen phosphate .2 Potassium chloride 1.15 Magnesium sulphate 7H O 0.65 Zinc sulphate 7H O 0.31 Manganese sulphate 411 0 0.068 Ferrous sulphate 7H O 0.125 Yeast extract 0.030

Tap water added to a volume of 1000 ml.

In order to keep the temperature in the fermenter constant at 30 C., water was circulated in an annulus constituted by the space between two concentric cylinders, he smaller one being the fermenter itself.

To the fermenter was added 14 litres of a 24 hour inoculum of Candida utilis grown on mixed C -C hydrocarbons containing normal parafiins; the cellular density in the fermenter was thus approximately 1 gram of Candida yeast (estimated at dry weight) per litre.

To the fermenter was added 100 grams per litre (of aqueous medium in the fermenter) of a heavy gas having the following characteristics:

Specific gravity (at 60 P): 0.8669 11 2 1.484

Cloud point: +13 C.

Pour point: +ll C.

Sulphur: 1.59% by wt.

11 paratfins: 16% by wt.

After an initial period of slow growth, lasting 6 hours, the cellular density was 2 grams/ litre; thereafter the rate of growth increased.

The temperature of the culture was kept at 30il C., pH at 4, the conditions of aeration and stirring being maintained to give 3 millimoles of per litre of medium per minute. Ammonia solution was admitted by an automatic pH controller.

When the cellular densty reached 4 grams/litre the fermenter was run continuously at a dilution rate of 0.1 vol./vol./hour. Meanwhile the amount of heavy gas oil in the fermenter was increased to 120 grams/litre. Broth was continuously withdrawn from the fermenter and subjected to decantation, 6% of spent medium being withdrawn and replaced by 65% of tap water.

To the upper phase was added 0.5 gram/litre of the non-ionic detergent sold under the trade name NI 29 and, after centrifuging, there were separately recovered:

Grams per litre Spent mineral medium 839' Non-metabolized gas oil 112 49 Paste of micro-organism The non-metabolized gas oil had the following characteristics The trace amount of detergent was found to have led to undesirable emulsifying characteristics in the gas oil.

This gas oil was tested for emulsion forming characteristics by the test method ASTM B.l400.01 56T. In thmis test is used 40 ccs. of water ,8 cc. of kerosine (as diluent) and 32 ccs. of the gas oil undergoing test. Results were shown in the following Table 2.

The recovered gas oil containing trace amounts of water and detergent was heated to 90 0, whereby the detergent was dissolved in the oil phase, and centrifuged at 90 C. in a Sharples tubular centrifuge at 14,000 g. to remove water. The gas oil, recovered in this centrifuging stage and containing trace amounts of surface active agent, was hydrogenated over a cobalt molybdate catalyst containing:

Percent by wt. M00 12.6 C00 2.45

200 ccs. of the catalyst was used in the form of 2.5 mm. x 2.5 mm. cylindrical pellets and hydrogenation conditions were:

Temperature-395 C.

Pressure-40 kg.

Gas oil throughput rate-3 vol./vol./ hour Hydrogen throughput rate-50 litres/hour The hydrogenation treatment did not change the pour point of the gas oil which remained at (minus) 6 C. Emulsion characteristics are shown in Table 2.

A micro-organism was grown on a heavy gas oil and harvested using the conditions of growth and harvesting described in Example 2, except that the micro-organism employed was of the strain Hansenula suaveolens in place of the Candida utilis (employed according to Example 2) and that the aqueous nutrient medium contained 0.300 gram/ litre of yeast extract.

The non-metabolized gas oil which was recovered from the broth had the following characteristics:

Sp gr. 60 (60 F.): 0.8820 n 1.492

Sulphur: 1.84% by wt. Cloud point: -l C. Pour point: 20 C. Water: Trace Detergent: Trace This gas oil was treated by hot centrifuging and hydrogenation under the conditions described in Example 2.

The emulsion characteristics of the foodstock, the first recovered gas oil and the hydrogenated gas oil, using the method of test described in Example 2 are shown in the following Table 3. Pour point remained constant at 20 C.

TABLE 3 Gas oil Recovered feedstock gas oil 1 Hydrogenated Emulsion test gas oil Separation time 30 sec. Gas oil (00.) 41. Water (00.) 38 Emulsion (00.) 1

60 min. 15 sec. 4 40 1 After fermentation.

The non-ionic detergent sold under the trade designation N1 29 is a product obtained by condensing a mixture of lauric alcohol and myristic alcohol with ethylene oxide, the product having an ethylene oxide chain of an average 8.5 units per terminal group.

What we claim is:

1. A process which comprises cultivating a micro-organism which is capable of growing on at least some straight chain hydrocarbons, cultivation being carried out in the presence of a hydrocarbon feedstock which consists in part of straight chain hydrocarbons in the presence of an aqueous nutrient medium and in the presence of a gas containing free oxygen and thereafter separating from the cultivated mixture, through the use of a surfactant, a fraction containing, as the major proportion thereof, unconsumed hydrocarbons together with a minor proportion of water and a minor proportion of surfactant, heating said fraction, whereby the surfactant is rendered partially or substantially water-insoluble, separating a hydrocarbon phase containing surfactant from an aqueous phase and hydrogenating said hydrocarbon phase under conditions such that the surfactant is rendered less effective or ineffective as an emulsifying agent.

2. A process according to claim 1 in which the feedstock is a fraction derived from petroleum.

3. A process according to claim 2 in which the feedstock is a gas oil.

4. A process according to claim 2 in which the feedstock is a kerosine.

5. A process according to claim 1 in which the microorganism is a yeast.

6. A process according to claim in which the microorganism is a straight chain paraffinic hydrocarbon-consuming yeast.

7. A process according to claim 6 in which the yeast is of the family Cryptococcaceae.

8. A process according to claim 7 in which the yeast is of the sub-family Cryptococcoideae.

9. A process according to claim 8 which the yeast is of the genus Torulopsis.

10. A process according to claim 8 in which the yeast is of the genus Candida.

11. A process according to claim 10 in which the yeast is Candida lipolytica.

12. A process according to claim 11 in which the yeast is Candida tropicalis.

13. A process according to claim 1 in which the microorganism is a bacterium.

14. A process according to claim 1 in which the surfactant is a non-ionic detergent.

15. A process according to claim 14 in which the nonionic detergent comprises, in the molecule, a chain of ethylene oxide groups.

16. A process according to claim 15 in which the detergent has the formula A-(CH CH O),,H where A is an alcohol residual group or acid residual group and where n is an integer.

17. A process according to claim 1 in which the fraction comprising unconsumed hydrocarbons together with a minor proportion of water and a minor proportion of surfactant is heated at a temperature in the range 100 C. and the product subjected to phase separation.

18. A process according to claim 1 in which the hydrocarbon phase, containing surfactant, is hydrogenated over a cobalt and molybdenum containing catalyst.

References Cited UNITED STATES PATENTS 6/1965 Champagnat 82 9/1966 Laine et al. 195-3 LIONEL M. SHAPIRO, Primary Examiner U.S. Cl. X.R. 195-3; 208-264 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION P t n No- 3,3,585 Dated October 27, 1970 Inventor(a) 1 3ernard Maurice Laine It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column Column Column Column Column Column Column Column line 33, "C.G.C. Murray" should read E.G.D.

Murray a line 37, "Cryptococcoaceae" should read Cryptococcaceae line 38, "Cryptococcocideae" should read Cryptococcoideae line 41, "Cryptococcocideae" should read Cryptococcoideae line 45, "Central" should read Centraal line 48, after "Paris" insert line 58, "Neurosporo" should read Neurospora line 70, "Micrococoaceae" should read Micrococcaceae Table l, opposite "Oil (cc. and under "Run 3", "48" should read 40 Table l, opposite "Water (cc.)" and under "Run 3", 3?) should read 38 line 58, "Diammonium hydrogen phosphate --.2" should read "Diammonium hydrogen phosphate---2.

Continued.

po-wso (5/69) Patent No Invontofls) Bernard Maurice Laine UNITED STATES PATENT OFFICE Dated October 27, 1970 PAGE 2 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column Column Column Column Column Column i Anew line 70, "he" should read the line 24, "67.," should read 657.

line 49, "thmis" should read this Table 2, opposite "Hydrogenated gas 39 Table 2, opposite "Hydrogenated gas 40 and Table 2, opposite "Hydrogenated gas "Oil (cc.)" and under oil", "30" should read "Water (cc.)" and under oil", "41" should read SEALED FEB-231971 Edward M. Fletcher, Ir. Mu Officer mum E. SGHUYLER, JR. Gomissionor o1 Patents 

